Detecting and identifying minute amounts of DNA or, after reverse transcription, RNA, from a sample is important for medical diagnostic purposes, routine care, research, etc. For example, the DNA may be from a bacteria or virus and indicate the presence and quantity thereof in the sample. One widely known method for detection and identification of minute amounts of DNA or RNA, miRNA, siRNA or other types of nucleic acid fragments, for example, is a polymerase chain reaction (hereinafter “PCR”). Through a series of cycles, the PCR exponentially amplifies or increases the amount of a specific target region of the nucleic acid, facilitating ease of analysis. Geneticists and medical researchers, for example, use PCR to determine if certain genes are present in a DNA sample and to create enough DNA to analyze the sequence. While PCR may be applied to DNA and, after potential additional process steps, RNA and other fragments of nucleic acids, hereinafter, it will be described with respect to DNA.
As known in the art, during a PCR procedure, a reagent or mastermix including a double strand of DNA, for example, may be divided into a plurality of wells on microtiter plates. The mastermix may include a suspension of ingredients, such as samples of DNA, selected DNA primer strands, DNA elements, enzymes, and fluorescent dye, for example. Other ingredients may be included in the mastermix. Multiple reactions typically occur in parallel on these plates and may detect multiple target regions of DNA simultaneously in the same well (multiplex). The reagent may be heated to denature, or separate into single strands, the target region of DNA. The reagent may then be cooled, allowing the primers to anneal or pair to their complementary sequence. The primers may be designed to bracket the target region of DNA to be amplified. The temperature may then be increased, allowing polymerase, such as Taq polymerase, for example, to attach at each priming site and extend (synthesize) a new DNA strand. This method may be repeated a number of times or cycles allowing the number of DNA strands to build up. As the method progresses through each cycle, the polymerase and primers may be reused, making copies of the DNA strands starting at the sequence where the primers bind. Because the sequence of the primers is known, the researcher may determine if the result of the PCR is positive or negative, or draw conclusions of the initial quantity of target DNA in the sample considered.
During standard PCR, the result of the PCR may be determined at the end of all the cycles by running the PCR mixture on a gel and visualizing the size of the DNA amplified, for example. During real-time PCR, the result of the PCR may be determined as the reaction progresses in real time. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that bind with any double-stranded DNA and, (2) sequence-specific DNA probes that are labeled with a fluorescent reporter dye, which permits detection only after hybridization of the probe with its complimentary DNA target region. With non-specific fluorescent dyes, the DNA-binding dye binds to all double-stranded DNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle. With the sequence-specific probes, the fluorescent reporter probe only detects the DNA containing the probe sequence; therefore, use of the reporter probe significantly increases specificity, and enables quantification even in the presence of non-specific DNA amplification. Fluorescent probes can be used in multiplex assays, for example, for detection of several target regions of DNA in the same reaction, based on specific probes with different-colored labels. Each color (detection/emission wave length) may be associated with a specific dye, and may be referred to as a dye. Amplification/detections devices may be equipped with different filters or other optical/electronic devices to separate different optical spectral ranges, i.e. the spectral range of the respective probe label. The number of target regions of DNA in the initial (unamplified) sample may be estimated by certain properties of the curve of detected fluorescence against cycle number, e.g., by comparison of the fluorescence intensity with a threshold intensity. In some instances the threshold intensity may be predefined, or may be based on the results at hand, for example. The cycle at which the fluorescence from a sample crosses the threshold is called the cycle threshold (Ct).
“Dye” and “channel” in this application are partly used interchangeably meaning that a certain dye can only be measured in one channel, or a one-to-one relationship exists, but, generally speaking, as a channel is typically a filter used to detect an emitted spectrum, different dyes can be measured in one channel like HEX, VIC JOE.
The inventors of the present invention have determined that existing problems with conventional real-time PCR specific target detection methods are:                The influence of reagent pipetting variations. For example, pipetting different amounts of sample into each of the wells in a single plate.        The variation that is observed when comparing the quantity of identical samples on different instruments; and        The variation or invalidation of the quantity due to specific non-optimal characteristics in the amplification/detection method, e.g. by stray light, temperature differences, differences from well to well in the plastics material (PCR plate and sealing) etc.        
These problems may cause systematically incorrect values for target DNA concentrations, which may lead to incorrect, disadvantageous diagnostic results, for example.
Two conventional ways to avoid at least some of the aforementioned problems are:                The use of adaptive algorithms to standardize and remove the background level, the result of which may be referred to as dR. The initial quantity of target region DNA is determined by comparing dR to a threshold value which may yield a Ct value (fractional cycle number for the point where fluorescence signal passes threshold). The threshold value is usually shared by all wells in the same channel on the same plate, or even pre-defined and used across multiple plates or even instruments. While this method adequately captures variations in reagent pipetting and some of the variations incurred by different instruments, the inventors of the present invention have determined that this method does not detect or eliminate at least some of the non-optimal features specific to this reaction run (e.g. cycle-dependent variation).        The use of normalization of the fluorescence signal of an actively reporting dye (a fluorescence color indicating a target DNA region) by the fluorescence of a passive or reference dye (another dye which is not used for target DNA region detection) in the same well, preferably per-cycle. After normalization, the background noise is estimated and removed or cleared. The resulting value is called the normalized intensity, or dRn. This method accounts for pipetting variations in reagents used (all reagents are at a constant ratio in the reagent mastermix, so if more is pipetted into a well of the plate, the ratio of the active and passive signal will remain unchanged), is relatively reproducible between different instruments, and may be able to detect and eliminate specific non-optimal characteristics of the amplification/detection run itself and thereby may produce reliable data.        
Conventional multiplex PCR includes multiple primer sets within a single PCR mixture to produce amplified regions of varying sizes that are specific to different DNA sequences. By targeting multiple regions of interest at once, additional information may be gained from a single test run that otherwise would require several times the reagents and/or more time to perform. However, in multiplex reaction runs involving multiple different dyes, the reaction run may be limited in the number of target DNA regions or regions of interest (ROI) that may be detected. In other words, in many PCR machines a limited number of dyes are available for each reaction run. For example, on the VERSANT kPCR platform offered by Siemens, only 5 dyes are available for quantitative detection of nucleic acids. With the normalization of the fluorescence signal of an actively reporting dye method described above, of these 5 dyes, one of the dyes is used as a passive reference, thereby limiting the number of separately detectable nucleic acids. For example, an HPV (human papilloma virus) assay may include 14 ROIs (plus a cellular control channel) that conventionally may be detectable by 4 dyes in a 5-dye system, as one dye is used as a reference dye. The assay, for example, may be as follows:                CY5: cellular control        HEX: HPV16        ALEXA: HPV18        FAM: HPV33, 35, 51, 52, 56, 31, 39, 68, 45, 58, 59        ROX: passive reference        There are presently not enough dyes to detect HPV45 on a separate channel; a signal reported in FAM channel could be due to any of the targets. Thus PCR for HPV45 needs to be run on a separate dye in a different reaction.        
It should be noted that while machines providing more than 5 dyes may be available for detecting more types of ROIs, as the number of dyes increases, the distinction between the different wavelengths/fluorescence decreases (e.g. by crosstalk), making identification of ROIs more difficult. Accordingly a need exists for an improved method and apparatus for detecting and determining the number of ROIs in a sample.